FR2959992A1 - PEPTIDES WITH ANTIPROTEASE ACTIVITY - Google Patents

Abstract

The present invention relates to a new family of peptides as defined in claim 1, as well as the RBM6 delta 6 protein, for their protease inhibitory activity. They can in particular be used for the therapeutic or prophylactic treatment of enterovirus, and in particular of a human rhinovirus such as RVH-2.

Description

The present invention relates to the general field of antivirals and more particularly, antiviral agents directed against enteroviruses. More specifically, the invention relates to novel peptides which exhibit a protease inhibitory activity (also called anti-protease activity), and in particular peptides which are of interest for the prophylactic or therapeutic treatment of Enterovirus infection. , and in particular by a rhinovirus or an echovirus. Enteroviruses are non-enveloped single-stranded RNA viruses of positive polarity. They belong to the Picornaviridae family.

Currently, different species of human enteroviruses have been identified: enterovirus A, enterovirus B (including Echoviruses including ECHOvirus 6), enterovirus C (with poliovirus), enterovirus D and human rhinovirus (RVH) A (comprising about 75 members including RVH-2), B (comprising about 25 members), C and D. These species are defined according to phylogenetic criteria based, in particular on that part of the genome encoding the structural proteins. Enteroviruses may be responsible for various types of pathologies, benign or severe, such as acute flaccid paralysis, aseptic meningitis, foot-hand-mouth syndrome, respiratory diseases, colds, otitis, sinusitis, acute or chronic heart disease, diarrhea, pancreatitis In particular, the clinical presentation of RVH is not limited to the common cold, since this virus is also associated with acute otitis media in children (Pitkaranta, Virolainen et al., 1998) and sinusitis. in adults (Pitkaranta, Arruda et al 1997). In addition, it can cause severe complications in at-risk subjects such as asthmatic patients (Nicholson, Kent et al., 1993) or those with immunodeficiency. No specific treatment against human rhinovirus is currently available and no vaccine exists to prevent this infection since nearly 100 different serotypes of RVH have been characterized. The main aim of treating the disease is to relieve the symptoms.

Different chemotherapeutic approaches have tried and are still trying to better combat RVH. These approaches can be classified into two groups and consist of: the group: (1) inhibit the virus undressing step by attaching different ligands at the capsid. Several synthetic compounds have been the subject of clinical trials in the United States: Disoxaril (WIN 51711), WIN 54954, Pleconaril (WIN 63843, picovir), Pirodavir (R 77975) and R 61837 (Patick, 2006). None of them have been approved by the US Food and Drug Administration. (2) prevent the attachment of the virus to its receptor. Nearly 90% of RVH serotypes use the ICAM-1 receptor to attach to susceptible cells. Thus, one of the strategies adopted is to block this fixation using soluble forms of ICAM-1 (sICAM-1) (Turner, Wecker et al 1999, Turner 2001). Tremacamra (BIRR-4), developed by Boechringer Ingelheim, has been evaluated in four double-blind clinical trials (Turner, Wecker et al., 1999). Tremacamra is capable of reducing the severity of experimentally induced colds whether administered before or after infection. This molecule seems, however, less effective when it is administered at variable times of 12 hours and more after infection. Moreover, it has been reported that a humoral response can develop against these soluble forms of ICAM-1 and thus reduce their effectiveness. (3) decrease the inflammatory response caused by RVH infection using interferons. Interferons are at the center of antiviral and anticancer responses via signaling cascades generated by their attachment to specific receptors. Several studies have evaluated the efficacy of recombinant interferon alpha 2b in the prevention or treatment of colds due to RVH (Hayden, Albrecht et al., 1986, Sperber, Levine et al., 1989). In these studies, intranasal injection of interferon has been shown to be effective in both "natural" and experimental colds, when done before infection. On the other hand, it has had little or no effect when administered to previously infected patients. Several side effects, such as bleeding from the nasal mucosa, have also been noted in these studies. 2nd group: inhibit the activity of viral proteases. The proteases 3C (named 3Cpro) and 2A (called 2Apro) of the rhinovirus are particularly interesting therapeutic targets. Indeed, they play an essential role in the replicative cycle of the virus: they catalyze the processing of the viral protein precursor and are at the center of the disturbances of certain intracellular functions induced by the infection with Rhinovirus. Different compounds have been tested for their ability to inhibit the activity of these proteases. Rupintrivir (AG7088) and Compound 1 for 3Cpro; elastinal, MPCMK (methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone), z-VAM.fmk and homophthalimide (LY353349) for 2Apro (Molla, Hellen et al., 1993; Wang, Johnson et al. 1998, Deszcz, Seipelt et al., 2004, Deszcz and Cencic et al., 2006). None of these compounds are currently used in therapy and no clinical trials including them have been described in the literature. Several studies have also been carried out on peptides derived from the sequence of the polyprotein of the RVH (US5559025, CA208221, EP0263214 and EP0263202 in particular) which exhibit a viral 2Apro inhibitory activity. Today, the therapeutic needs for the treatment of Rhinovirus-related conditions are not met. Also, in this context, the present invention proposes to provide new peptide derivatives, useful for the treatment of an enterovirus infection, and in particular by a human rhinovirus. The subject of the present invention is therefore peptides whose C-terminal portion has the sequence X1-X2-X3-X4-X5-X6 in which: X1 and X3, which are identical or different, are Leu, Ile or Phe, or Unconventional amino acid derived from Leu, Ile or Phe, X2 and X4, the same or different, are any amino acid, X5 is Asn, Thr or Gln, or an unconventional amino acid derived from Asn, Thr or Gln, and X6 is a hydrophobic amino acid. The subject of the present invention is also the chemical derivatives of such peptides. The peptides according to the invention have a C-terminal X1-X2-X3-X4-X5-X6 peptide sequence as defined above which corresponds to a pseudo-substrate of the 2Apro of the RVH-2 and which mimics the P1 sequence. -P6 (according to the nomenclature proposed by Schechter and Berger (Schechter and Berger 1968) of the half-site of cleavage of certain natural substrates of 2Apro.In the context of the invention, the inventors have demonstrated that this motif is directly involved in the interaction with 2Apro of RVH-2 In addition, the inventors have demonstrated that the peptides according to the invention have a broader spectrum of action that extends to other 2Apro enteroviruses, and therefore more widely to 2Apro enteroviruses, and possibly any protease, viral or non-viral, in particular having the same catalytic properties, because an in vitro interaction between these peptides, and in particular with the peptide LVLQTM (SEQ ID No. 9) of the C-terminal end of the protein Cellular RBM6 delta 6 and 2Apro RVH-2 were highlighted. On the other hand, it has been demonstrated that such peptides behave as reversible inhibitors of the viral enzyme in vitro as well as in mammalian cells. In the same way, it has been demonstrated that the C-terminal end of RBM6 delta 6 also has inhibitory properties on 2Apro of ECHOvirus 6, which makes it possible to envisage the peptides according to the invention as inhibitors of 2Apro. enteroviral. The invention also relates to the peptides whose C-terminal part corresponds to the sequence: X1-X2-X3-X4-X5-X6 previously defined, as well as those which will be defined in the remainder of the description, as well as the RBM6 delta 6 protein in isolated form, which exhibit an inhibitory activity of a viral or cellular protease, in particular an inhibitory activity of the protease 2A of rhinovirus and / or a protease (viral or

non-viral) having, in particular, the same catalytic properties as protease 2A rhinovirus. According to another of its aspects, the subject of the invention is also the RBM6 delta 6 protein in an isolated form and the peptides as defined above, as well as those which will be defined in the remainder of the description, used for the therapeutic treatment or prophylactic enterovirus, as well as pharmaceutical compositions comprising them, in association with a pharmaceutically acceptable excipient.

Before giving a more detailed description of the invention, a number of definitions of the terms used to define the invention will be recalled. By "peptide" is meant any sequence of amino acids, without greater limitation of size. The term peptide therefore encompasses large peptides, classically referred to as proteins. The term "peptide" refers to a sequence of two or more amino acids linked together by peptide bonds or modified peptide bonds. The amino acids present in the peptides according to the invention, unless otherwise specified, may be natural amino acids: Leucine (Leu), Glycine (Gly), Alanine (Ala), Isoleucine (Ile ), Valine (val), Serine (Ser) Threonine (Thr), Phenylalanine (Phe), Tryptophan (Trp), Tyrosine (Tyr), Proline (pro), Cysteine (Cys), Methionine (Met), Asparagine (Asn) , Glutamine (Gin), Arginine (Arg), Lysine (Lys), Histidine (His), Aspartic Acid (Asp), Glutamic acid (Glu) which are of L conformation, or non-natural amino acids, named amino acids not conventional ones such as especially the amino acids below Nle = L-norleucine; Aabu = α-aminobutyric acid; Hphe = L-homophenylalanine; Nva = L-norvaline; Gabu = y-aminobutyric acid; Daia = D-alanine; Dcys = D-cysteine; Dasp = D-aspartic acid; Dglu = D-glutamic acid; Dphe = D-phenylalanine; Dhis = D-histidine; Dile = D-isoleucine; Dlys = D-lysine; God = D-leucine; Dmet = D-methionine; Dasn = D-asparagine; Dpro = D-proline; Dgln = D-glutamine; Darg = D-arginine; Dser = D-serine; Dthr = D-threonine; Dval = D-valine; Dtrp = D-tryptophan; Dtyr = D-tyrosine; Dorn = D-ornithine; Aib = aminoisobutyric acid; Etg = L-ethylglycine; Tbug = L-t-butylglycine; Pen = penicillamine; Anap = α-naphthylalanine; Chexa = cyclohexylalanine; Cpen = cyclopentylalanine; Cpro = aminocyclopropane carboxylate; Norb = aminonorbornylcarboxylate; Mala = L-α-methylalanine; Mcys = L-amethylcysteine; Masp = L-α-methylaspartic acid; Mglu = L-amethylglutamic acid; Mphe = L-α-methylphenylalanine; Mhis = L-amethylhistidine; Mile = L-α-methylisoleucine; Mlys = L-α-methyllysine; Mleu = L-α-methylleucine; Mmet = L-α-methylmethionine; Masn = L-amylasparagine; Mpro = L-α-methylproline; MgIn = L-α-methylglutamine; Marg = L-α-methylarginine; Mser = L-α-methylserine; Mthr = L-amethylthreonine; Mval = L-α-methylvaline; Mtrp = L-α-methyltryptophan; Mtyr = L-α-methyltyrosine; Morn = L-α-methylornithine; Mnle = L-methylnorleucine; Maabu = α-amino-α-methylbutyric acid; Mnva = L-amethylnorvaline; Mhphe = L-α-methylhomophenylalanine; Metg = L-amethylethylglycine; Mgabu = α-methyl-γ-aminobutyric acid; Maib = α-methyliaminoisobutyric acid; Mtbug = L-α-methyl-4-butylglycine; Mpen = α-methylpenicillamine; Manap = α-methyl-α-naphthylalanine; Mchexa = α-methylcyclohexylalanine; Mcpen = a-methylcyclopentylalanine; Dmala = D-α-methylaianine; Dmorn = D-a-methylornithine; Dmcys = D-amethylcysteine; Dmasp = α-methylaspartic acid; Dmglu = D-amethylglutamic acid; Dmphe = D-α-methylphenylalanine; Dmhis = D-amethylhistidine; Dmile = D-α-methylisoleucine; Dmlys = D-α-methyllysine; Dmleu = D-α-methylleucine; Dmmet = D-a-methylmethionine; Dmasn = D-α-methylasparagine; Dmpro = D-a-methylproline; Dmgln = D-amethylglutamine; Dmarg = D-α-methylarginine; Dmser = D-α-methylserine; Dmthr = D-α-methylthreonine; Dmval = D-a-methylvaline; Dmtrp = D-amethyltryptophan; Dmtyr = D-α-methyltyrosine; Nmala = L-N-methylalanine; Nmcys = L-N-methylcysteine Nmasp = L-N-methylaspartic acid; Nmglu = L-N-methylglutamic acid; Nmphe = L-N-methylphenylalanine; Nmhis = L-N-methylhistidine; Nmie = L-N-methylisoleucine; Nmlys = L-N-methyllysine; Nmleu = L-N-methylleucine; Nmmet = L-N-methylmethionine; Nmasn = L-N-methylasparagine; Nmchexa = N-methylcyclohexylalanine; Nmgln = L-N-methylglutamine; Nmarg = L-N-methylarginine; Nmser = L-N-methylserine; Nmthr = L-N-methylthreonine; Nmval = L-N-methylvaline; Nmtrp = L-N-methyltryptophan; Nmtyr = L-N-methyltyrosine; Nmorn = L-N-methylornithine; Nmne = L-N-methylnorleucine Nmaabu = N-amino-methylbutyric acid; Nmnva = L-N-methylnorvaline; Nmhphe = L-N-methylhomophenylalanine; Nmetg = L-N-methylethylglycine; Nmgabu = N-methyl-y-aminobutyric acid; Nmcpen = N-methylcyclopentylalanine; Nmtbug = L-N-methyl-t-butylglycine; Nmpen = N-methylpenicillamine; Nmanap = N-methyl-α-naphthylalanine; Nmaib = N-methylaminoisobutyric acid; Dnmala = D-N-methylalanine; Dnmorn = D-N-methylornithine; Dnmcys = D-N-methylcysteine; Dnmasp = D-N-methylaspartic acid; Dnmglu = D-N-methylglutamic acid; Dnmphe = D-N-methylphenylalanine; Dnmhis = D-N-methylhistidine; Dnmile = D-N-methylisoleucine; Dnmlys = D-N-methyllysine; Dnmleu = D-N-methylleucine; Dnmmet = D-N-methylmethionine; Dnmasn = D-N-methylasparagine; Dnmpro = D-N-methylproline; Dnmgln = D-N-methylglutamine; Dnmarg = D-N-methylarginine; Dnmser = D-N-methylserine; Dnmthr = D-N-methylthreonine; Dnmval = D-N-methylvaline; Dnmtrp = D-N-methyltryptophan; Dnmthr = D-N-methyltyrosine; Nala = N-methylglycine (sarcosine); Nasp = N- (carboxymethyl) glycine; Nglu = N- (2-carboxyethyl) glycine; Nphe = N-benzylglycine; Nhhis = N- (imidazolylethyl) glycine; N1 = N- (1-methylpropyl) glycine; Nlys = N- (4-aminobutyl) glycine; Nleu = N- (2-methylpropyl) glycine; Nmet = N- (2-methylthioethyl) glycine; Nhser = N- (hydroxyethyl) glycine; Nasn = N- (carbamylmethyl) glycine; Ngln = N- (2 carbamylethyl) glycine; Nval = N- (1-methylethyl) glycine; Narg = N- (3-guanidinopropyl) glycine; Nhtrp = N- (3-indolylethyl) glycine; Nhtyr = N- (phydroxyphenethyl) glycine; Nthr = N- (1-hydroxyethyl) glycine; Ncys = N- (thiomethyl) glycine; and N- (3-aminopropyl) glycine; Ncpro = N-cyclopropylglycine; Ncbut = N-cyclobutyglycine; Nchex = N-cyclohexylglycine; Nchep = N-cycloheptylglycine; Nococt = N-cyclooctylglycine; Ncdec = N-cyclodecylglycine; Ncund = N-cycloundecylglycine; Ncdod = N-cyclododecylglycine; Nbhm = N- (2,2-diphenylethyl) glycine; Nbhe = N- (3,3-diphenylpropyl) glycine, Nnbhm = N- (N- (2,2-diphenylethyl) carbamylmethyl) glycine Nnbhe = N- (N- (3,3-diphenylpropyl) carbamylmethyl) glycine; Nbmc = 1-carboxy-1- (2,2-diphenylethylamino) cyclopropane; and Naeg = N- (2-aminoethyl) glycine. In the context of the invention, the general term "amino acid" thus refers to natural amino acids and non-natural amino acids, called unconventional amino acids. By "unconventional amino acid derived from a natural amino acid" is meant an amino acid belonging to the above list whose name includes the name of the natural amino acid from which it is derived. The term "chemical derivative" of the peptides according to the invention is understood to mean peptides which comprise one or more additional groups which are not naturally present on peptide sequences. Such groups have, for example, the function of improving their solubility or half-life in a biological medium (such as, for example, the group Z -C (O) OBz with Bz = benzyl which allows the peptide to be better to cross the plasma membrane of the cells), or to reduce their toxicity or undesirable side effects, or to form an irreversible covalent bond with their target protein (such as, for example, the group fmk 25 = fluoromethylketone which can form a link covalent with a catalytic cysteine of the active center of an enzyme) in particular to perform crystallographic studies. Also, in order to improve their resistance to degradation in vivo, the peptides according to the invention can be in protected form. In view of the application of the peptides according to the invention, the form of protection must obviously be a biologically compatible form and should preferably be compatible with prophylactic and therapeutic use. Many biologically compatible forms of protection, well known to those skilled in the art, can be envisaged. By way of example, mention may be made of acylation or acetylation of the amino-terminal end, or amidation or esterification of the carboxy-terminal end of the peptide. Thus, the peptides according to the invention may be substituted on their N-terminal end with an acetyl group, a benzoyl group, a tosyl group or a benzyloxycarbonyl (Z) group, or on their C-terminus by amidation of the hydroxyl function. -COOH group by an NYY group with Y which represents an alkyl chain, especially from 1 to 4 carbon atoms, or by esterification with an alkyl group, especially from 1 to 4 carbon atoms. It is also possible that both ends of the peptide are protected. By "purified" or "isolated" peptide or protein is meant that the peptide or protein in question is obtained in a form substantially free of macromolecules of the same type. That is to say, preferably, the peptide or the protein in question represents at least 75% by weight, preferably at least 85% by weight, and more preferably at least 95% by weight, or even more than 98% by weight. mass of biological macromolecules present of the same type (but water, buffers or molecules of low molecular weight, especially less than 1000 daltons may be present). Purification methods are known to those skilled in the art. A naturally occurring protein may, for example, be purified from lysates and cell extracts, from the supernatant of the culture medium, by methods used individually or in combination, such as fractionation, chromatography methods, immunoaffinity using specific mono or polyclonal antibodies, etc. Within the scope of the invention, the peptides may be any size, but include at least the 6 amino acids X1-X2-X3-X4-X5 X6 (NH2-> COOH) as defined within the scope of the invention. When more than 6 amino acids are present in the peptides according to the invention, the binding to the XI-X2-X3-X4-X5-X6 sequence is via X1. Each of the amino acids present in the peptides according to the invention may be a natural or unconventional amino acid. The peptides according to the invention comprise, for example, from 6 to 1000 amino acids. Nevertheless, when the peptides according to the invention will have to be administered in a pharmaceutical composition, depending on the nature of this composition, it may be advantageous that their size does not exceed 50 or 20 amino acids. According to a particular embodiment, these peptides will correspond to artificial peptides. That is, they will not match the entire sequence of a biological protein. By "biological protein" is meant any protein naturally occurring and naturally produced by a living organism, and especially the RBM6 delta 6 protein of sequence: MWGDSRPANR TGPFRGSQEE RFAPGWNRDY PPPPLKSHAQ ERHSGNFPGR DSLPFDFQGH SGPPFANVEE HSFSYGARDG PHGDYRGGEG PGHDFRGGDF SSSDFQSRDS SQLDFRGRDI HSGDFRDREG PPMDYRGGDG TSMDYRGREA PHMNYRDRDA HAVDFRGRDA PPSDFRGRGT YDLDFRGRDG SHADFRGRDL SDLDFRAREQ SRSDFRNRDV SDLDFRDKDG TQVDFRGRGS GTTDLDFRDR DTPHSDFRGR HRSRTDQDFR GREMGSCMEF KDREMPPVDP NILDYIQPST QDREHSGMNV NRREESTHDH TIERPAFGIQ KGEFEHSETR EGETQGVAFE HESPADFQNS QSPVQDQDKS QLSGREEQSS DAGLFKEEGG LDFLGRQDTD YRSMEYRDVD HRLPGSQMFG YGQSKSFPEG KTARDAQRDL QDQDYRTGPS EEKPSRLIRL SGVPEDATKE EILNAFRTPD GMPVKNLQLK EYNTGNSNDP GQRSYPGVCI KPGFLVLQTM (SEQ ID NO: 1), the DNA sequence has been submitted October 9, 2007 in GenBank and has the reference F1156542). By "inhibitory activity of a protease" is meant a minimum inhibitory activity of 50% on at least one reference test for measuring the inhibitory activity on said protease. For the protease 2A of Rhinovirus 2, a hydrolysis test of the calorimetric substrate TRPIITTA (SEQ ID No. 18) -pNa may be taken as a reference test, and in particular the test described in the examples below.

The term "treatment" means any prophylactic or suppressive therapeutic measure of a disease or disorder leading to a desirable clinical effect or any beneficial effect, including in particular the suppression or decrease of one or more symptoms, regression, slowing or cessation of the progression of the virus or the disorder associated with it. By "therapeutically effective amount" is meant any amount of a composition that improves one or more of the characteristic parameters of the viral condition being treated. Some embodiments of the invention will now be described in more detail. The invention particularly relates to more specific families of peptides as defined above. In particular, the invention relates to the peptides which correspond to the X1-X2-X3-X4-X5-X6 sequence as defined above, as well as the chemical derivatives of such peptides. For these different peptides, those whose C-terminal part corresponds to the X1-X2-X3-X4-X5-X6 sequence as defined above, or those which correspond exactly to this sequence, X6 is, for example, an acid amine selected from Leu, Cys, Val, Met, Pro, Tyr, Ile, Ala, Phe, Trp and unconventional amino acids derived from Leu, Cys, Val, Met, Pro, Tyr, Ile, Ala, Phe and Trp. In particular, X6 is an amino acid selected from Leu, Cys, Val, Met, and is preferably Met. In fact, the presence of a methionine in such a position favors the interaction of the peptide with the targeted protease. This has been demonstrated in the publication of (Deszcz, Cencic et al., 2006), for the 2Apro protease of the RVH where the compound zVAD.fmk, a caspase inhibitor capable of inhibiting both the activity of the 2Apro of the in vitro RVH and CBV4 and replication of these viruses, was modified by addition of a methyl ester group on aspartate (aspartate residue replaced by a methionine) in position P1 in zVAD.fmk and allowed to limit the action of this compound on proteases 2A and, thus, increase its specificity with respect to viral proteases. The nature of X2 and X4 is not critical: for example, the same or different X2 and X4 are an amino acid selected from Val, Lys, Sern Thr, Arg, Cys, His, Phe, Tyr and Gln and the acids unconventional amines derived from these.

Peptides whose C-terminal part corresponds to a hexapeptide chosen from: IKLVTL (SEQ ID NO: 2), LSLVTL (SEQ ID NO: 3), FRLTTL (SEQ ID NO: 4), LCLHTC (SEQ ID NO: 5) , LFLYTC (SEQ ID NO: 6), LYIYTV (SEQ ID NO: 7), LKLQTV (SEQ ID NO: 8), LVLQTM (SEQ ID NO: 9), LRLKNL (SEQ ID NO: 10), LRLRNL (SEQ ID NO. No. 11), as well as the peptides which exactly correspond to a hexapeptide chosen from IKLVTL (SEQ ID No. 2), LSLVTL (SEQ ID No. 3), FRLTTL (SEQ ID No. 4), LCLHTC (SEQ ID No. 5), LFLYTC (SEQ ID NO: 6), LYIYTV (SEQ ID NO: 7), LKLQTV (SEQ ID NO: 8), LVLQTM (SEQ ID NO: 9), LRLKNL (SEQ ID NO: 10), LRLRNL ( SEQ ID NO: 11), as well as their chemical derivatives, are particular examples of peptides according to the invention. The hexapeptide LVLQTM (SEQ ID No. 9), detected at the C-terminal end of the RBM6 delta 6 protein, as well as its chemical derivatives, is particularly preferred. The peptides according to the invention can be obtained by proteolysis or synthetically, chemically. In particular, the peptides according to the invention can be obtained by solid phase or liquid homogeneous chemical synthesis, or by enzymatic synthesis, or by genetic engineering, according to techniques well known to those skilled in the art. The peptides according to the invention, but also the RBM6 delta 6 protein whose biological function had not yet been identified, are particularly interesting from the prophylactic and therapeutic standpoints, for the treatment of Enteroviruses, in mammals and, in particular, in humans, and in particular human rhinoviruses, such as RVH-2, echoviruses such as human ECHO-6, or polioviruses and coxsackieviruses whose enteroviral protease 2A has strong homologies with the 2Apro of ECHO 6 whose activity is also inhibited by the LVLQTM motif (SEQ ID No. 9). The peptides according to the invention and the isolated RBM6 delta 6 protein can therefore be used as a medicament for the treatment of colds, otitis, sinusitis, or acute flaccid paralysis, aseptic meningitis, hand-foot-and-mouth syndrome, respiratory diseases. , acute or chronic heart disease, diarrhea, pancreatitis, ocular involvement, encephalitis.

More generally, the peptides according to the invention and the isolated RBM6 delta 6 protein may be used to inhibit a viral protease or a cellular protease such as elastase, chymotrypsin-like proteases and caspases (Skern, Sommergruber et al., 1991 ). In fact, enterovirus protease 2A is a cysteine protease whose activity can be modulated by known inhibitors of cellular proteases. Thus, the P2A of RVH-14 and type 1 poliovirus is inhibited by specific elastase inhibitors (Molla, Hellen et al., 1993). Of course, the peptides according to the invention and the isolated RBM6 delta 6 protein may be used to inhibit viral or non-viral proteases, other than Enterovirus 2Apro, if these proteases have catalytic properties close to viral 2Apro. One of the applications is the use of a peptide according to the invention or of the RBM6 delta 6 protein in the preparation of cell lysates, in particular to supplement cocktails of protease inhibitors.

Nevertheless, according to one of its essential aspects, the invention relates to the peptides according to the invention, as well as the isolated RBM6 delta 6 protein, for their use as medicaments, as well as the pharmaceutical compositions containing one of the peptides according to the invention. invention or the isolated RBM6 delta 6 protein, in combination with at least one suitable pharmaceutical excipient. By suitable or acceptable pharmaceutical excipient is meant any compound or vehicle entering a pharmaceutical composition which does not cause or almost no side reactions and which makes it possible, for example, to facilitate the administration of the peptides according to the invention, to increase their life and / or effectiveness in the body, increase their solubility, or improve their conservation. These pharmaceutically acceptable excipients are well known to those skilled in the art and will be adapted by the latter, depending on the nature and mode of administration of the peptides according to the invention. The peptide may, for example, be solubilized in one or more pharmaceutically acceptable solvents, conventionally used by those skilled in the art, such as, for example, water, glycerol, ethanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols, petrolatum, a vegetable oil and any mixture of these solvents. The peptide may also be formulated in a pharmaceutical vector, such as liposomes, or adsorbed onto powdery organic polymers, inorganic carriers such as talcs and bentonites, and more generally solubilized in, or attached to, any pharmaceutically acceptable carrier. The peptides according to the invention may, for example, be administered orally, sublingually, subcutaneously, intramuscularly, intravenously, topically, intratracheally, intranasally, transdermally, rectally or intraocularly.

Their modes of administration, dosages and optimal dosage forms will be determined according to the criteria generally taken into account in the establishment of a treatment adapted to a patient such as, for example, the age or body weight of the patient, the severity of his condition. general, tolerance to treatment and the observed side effects. The peptide will be present within the composition in a therapeutically effective amount. For example, a peptide according to the invention may be present in the compositions of the invention at a concentration of between approximately 0.0005 and 500 ppm (parts per million, weight / weight), and preferably at a concentration of between 0, About 1 and 5 ppm based on the total weight of the final composition. A peptide according to the invention may be used alone or in combination with at least one other active agent, in a pharmaceutical composition. The peptides according to the invention may in particular be used in combination with an already known protease inhibitor and / or with a compound under evaluation. The invention therefore also relates to pharmaceutical compositions containing such combinations. Other aspects and advantages of the invention will be better understood from the following examples which illustrate the invention, but are not limiting in nature. These examples refer to the appended figures: FIG. 1 shows an alignment of the C-terminal ends of the peptides identified in double hybrid. The common sequence unit L-X-L-XT / N-Z (where X is any amino acid and Z is a hydrophobic amino acid) is shown and aligned with the P6-P1 residues of various known cleavage sites of 2Apro. Figure 2 shows the results of so-called "pull-down" experiments conducted in the presence of 2Apro of RVH-2 and peptide LVLQTM (SEQ ID No. 9). Bacterial lysates containing the Sreptag-SUMO, Stretag-SUMO-SEQ ID No. 9 or RVH-2 2Apro- (His) 6 proteins were mixed in pairs and subjected to affinity chromatography on magnetic beads coated with strep- Tactin. The fixed complexes were separated on SDS-PAGE acrylamide gel and stained with Coomassie Blue. lane 1: Streptag-SUMO and RVH-2 2Apro- (His) 6 lane 2: Streptag-SUMO-LVLQTM and RVH-2 2Apro- (His) 6 lane 3: 2Apro- (His) 6 of RVH-2 purified on beads Nickel. Figures 3A and 3B show replenishment experiments of a complete cleavage site of 2Apro of RVH-2 or ECHO 6 from the LVLQTM peptide (SEQ ID NO: 9). Recombinant luciferases (61 kDa) containing the hybrid sites LVLQTM-GPSDM (SEQ ID No. 14) or PIIITTAGPSDM (SEQ ID No. 16) were synthesized in an in vitro transcription / translation system in the presence of 35S-methionine and incubated with recombinant 2Apro- (His) 6 of RVH-2 (FIG. 3A) or 2Apro- (His) 6 of ECHO 6 (FIG. 3B). Samples of the incubation medium were taken at times 15, 30, 45 and 60 minutes and the proteins were separated by SDS-PAGE and analyzed by fluorography. Figure 4 shows the percent inhibition of StrepTag-SUMO-SEQ ID NO: 9 peptide on the cleavage activity of 2Apro of RVH-2 in vitro. The cleavage of the colorimetric substrate TRPIITTA (SEQ ID NO: 18) -p-nitroanilide (25 μM) specific for 2Apro of RVH-2 was measured in vitro in the presence of different concentrations of the Streptag-SUMO protein, whether or not containing the peptide. LVLQTM (SEQ ID NO: 9). Absorbance measurements were made continuously at 405 nm for 10 minutes and the initial rate of each reaction was determined. The percent inhibition for a given concentration of Streptag-SUMO protein or StrepTag-SUMO-SEQ ID NO: 9 was calculated by reporting the determined initial reaction rates in the presence and absence of the protein. The percent inhibition values given are the average of the values obtained from 3 independent experiments. Standard deviations are also represented.

Figure 5 shows the cellulo inhibition of the RVH-2 2Apro eIF-4Gase activity by the LVLQTM peptide (SEQ ID NO: 9). Cleavage of eIF-4G cell factor (220 kDa) was studied in A549 cells overexpressing RBM6 delta 6274-520 and / or RVH-2 2Apro for 48h. Protein extracts were prepared, analyzed by SDS-PAGE and transferred to nitrocellulose membrane. The samples were then incubated with a rabbit anti-eIF-4G polyclonal antibody and then a peroxidase-coupled secondary anti-rabbit antibody. The control corresponds to A549 cells placed in the presence of the transfection product alone. Figure 6 shows the inhibition of RVH-2 infection in A549 cells overexpressing RBM6 delta 6274-520. Transient overexpressing A549 cells RBM6 delta 6274-520, delta RBM6 6274-514 or SUMO protein used as a negative control were infected with RVH-2 at a MOI of 1 for 20h. The antiviral effect was determined by the measurement of TCID50 (infectious dose 50% in sensitive tissue culture) 1) Identification of partners of the protease 2A of RVH-2 by the double-hybrid technique in yeast, in order to identify antiviral inhibitors targeting this enzyme The LexA-RVH-2 2Apro fusion protein was used as a bait to screen a human placenta cDNA library. For this purpose, the sequence encoding P2A of the RVH-2 (MGPSDMYVHVGNLIYRNLHLFNSEMHESILVSYSSDLIIYRTNTVGDDYIPSCDCTQ ATYYCKHKNRYFPITVTSHDWYEIQESEYYPKHIQYNLLIGEGPCEPGDCGGKLLCKH GVIGIVTAGGDNAFIDLRHFHCAEEQ, SEQ ID NO: 12) was synthesized by optimizing the codon usage in yeast and cloned into the vector PB27 molten LexA. This construct was sequenced and used as a bait to screen a human placenta cDNA library inserted into the P6 vector (Vojtek and Hollenberg 1995). High throughput screening of 53.1 million clones according to the approach described by Fromont-Racine et al. (Fromont-Racine, Rain et al 1997) identified 51 potential partners. For each of them, the cDNA was amplified by PCR and sequenced at the 5 'and 3' ends to identify the corresponding gene in the GenBank database (NCBI). A PBS (Predicted Biological Score) confidence index was assigned to each of the identified interactions (Formstecher, Aresta et al., 2005). This index reflects the biological reality of the interactions detected by this technique (Rain, Selig and others 2001, Wojcik, Boneca et al., 2002). Of the 51 isolated clones, two had significant PBS. The latter coded for the C-terminal end (residues 274 to 520) of the RBM6 delta 6 cell protein (GenBank: F1156542) that results from the alternative splicing of the RBM6 pre-mRNA (Timmer, Terpstra et al., 1999). The alignment of the C-terminus of each of these 51 peptides made it possible to isolate 10 different ones (one of which corresponded to the end of RBM6 delta 6, LVLQTM, SEQ ID No. 9) which had in common at their C-terminus, the peptide unit X1-X2-X3-X4-X5-X6 in which - X1 and X3, which are identical or different, are Leu or Ile, - X2 and X4, which are identical or different, are any amino acid, X5 is Asn or Thr, and X6 is a hydrophobic amino acid, as are the chemical derivatives of such peptides.

The peptides are IKLVTL (SEQ ID NO: 2), LSLVTL (SEQ ID NO: 3), FRLTTL (SEQ ID NO: 4), LCLHTC (SEQ ID NO: 5), LFLYTC (SEQ ID NO: 6). , LYIYTV (SEQ ID NO: 7), LKLQTV (SEQ ID NO: 8), LVLQTM (SEQ ID NO: 9), LRLKNL (SEQ ID NO: 10) and LRLRNL (SEQ ID NO: 11). Figure 1 shows the complete peptide sequences of isolated clones comprising said peptides.

In the international nomenclature, the cleavage site between residues P1 and Pl 'of a protease substrate is represented as follows (Schechter and Berger 1968): NH2-P5-P4-P3-P2-P1-P1- P2'-P3'-P4'-P5'-000H.

The X1-X2-X3-X4-X5-X6 motif as defined in the context of the invention partially mimics the P4-P1 cleavage half-site presented in FIG. 1 (p4LXTXp1) of several natural substrates known to enterovirus 2Apro. . In addition, the motifs p3QTMp1 and p4LQTp2 of RBM6 delta 6 are identical to those found at the same positions at the RVH-2 2Apro cleavage sites of PABP1 (Poly A Binding Protein) and the VP1-2Apro junction of the polyprotein of RVH-62 respectively, as shown in Figure 1. For the continuation of the study, the peptide LVLQTM derived from RBM6 delta 6, SEQ ID No. 9 (C-terminal end of RBM6 delta 6) was selected because it has a methionine in P1, a residue described elsewhere to significantly enhance the interaction with 2Apro of RVH-2 (Sommergruber, Ahorn et al., 1992).

2) The peptide LVLQTM, SEQ ID No. 9 of the protein RBM6 delta 6 interacts in vitro with the protease 2A of the R VH-2 The peptide LVLQTM, SEQ ID No. 9 of RBM6 delta 6 was chosen in order to validate in vitro in "pull-down" experiments, the results obtained previously in vivo in yeast. The RVH-2 protease 2A is cloned into the overexpression vector pSCodon1.2 (Eurogentec) between the NdeI and XhoI restriction sites and melt with a C-terminal side (His) tag. The nucleotide sequence coding for the LVLQTM motif, SEQ ID No. 9, is cloned into the pET-SUMO vector (Invitrogen), between two BsaI restriction sites, in phase at the C-terminus of the SUMO protein. The Streptag WSHPQFEK tag (SEQ ID NO: 13) is added on the N-terminal side of the SUMO-SEQ ID No. 9 fusion protein. This construct is transferred into the vector pSCodon1.2. Similarly, a pSCodon1.2 [streptag-SUMO] plasmid is constructed to serve as a control in the experiments described hereinafter.

These plasmids are used to transform the E.coli B strain SE1 [F-, CmR, ompT, Ion, hsdSB (rB-, mB-), gal, dcm, DE3 (lacI, T7polymerase under the control of the promoter PIacUV5) , ccdB +] and produce the proteins expressed by each of these constructs. For this, the bacteria are cultured in a self-induction medium ZYP-5052 (Studier 2005) at 37 ° C for 5-6 hours with stirring. They are then transferred to 20 ° C for 16 to 18 hours. The cells are lysed with a mixture of detergents (BugBuster, Novagen). The lysates thus obtained are collected in pairs (StrepTag-SUMO-SEQ ID No. 9 and RVH-2 2Apro- (His) 6 or StrepTag-SUMO and RVH-2 2Apro- (His) 6) and subjected to chromatography. of affinity on magnetic beads coated with Strep-Tactin (Qiagen) for one hour. Protein complexes attached to the beads after several washes are separated on SDS-PAGE acrylamide gel and stained with Coomassie Brillant Blue R250.

In this way, it has been demonstrated that RVH-2 2Apro- (His) 6 co-eluted with StrepTag-SUMO-SEQ ID No. 9 but not with StrepTag-SUMO, as shown in Figure 2, by comparing Streptag tracks. -SUMO + RVH-2 2Apro and Streptag-SUMO-LVLQTM + RVH-2 2Apro. Thus, the SEQ ID No. 9 motif is necessary and sufficient to interact in vitro with the protease 2A of the RVH-2, which confirms the results obtained in double-hybrid.

3) Reconstitution of a Complete Cleavage Site of the 2Apro of RVH-2 from the Peptide SEQ ID No. 9 The Assumption that the Peptide LVLQTM, SEQ ID No. 9 mimics a half-cleavage site of substrates known to RVH-2 protease 2A was verified in a series of experiments where a hybrid site LVLQTM-GPSDM (SEQ ID NO: 14) was synthesized. This site contains at the P6-P1 positions the peptide LVLQTM (SEQ ID No. 9) derived from RBM6 delta 6 and in positions P1'-P5 'the sequence GPSDM (SEQ ID No. 15) corresponding to the residues found in the cleavage site VP1-P2A (PIITTA-GPSDM, SEQ ID No. 16) of the 2Apro of the RVH-2 polyprotein.

The nucleotide sequences coding for LVLQTM-GPSDM (SEQ ID No. 14) or PIITTA-GPSDM (SEQ ID No. 16) are inserted between the NheI and BglII sites of a nucleotide sequence coding for a peptide linking the N and C domains. terminals of a recombinant luciferase in the pGloSensorTM-10F vector (Promega). The corresponding proteins are synthesized in an in vitro transcription / translation system in the presence of [35S] methionine (TNT SP6 High Yield Wheat Germ Master Mix, Promega). The whole is incubated for 2 hours at 25 ° C. In parallel, the protease 2A of the RVH-2 is cloned in the vector pSCodon1.2 (Eurogentec) between the NdeI and XhoI restriction sites in fusion with a label (His) 6 on the C-terminal side. This plasmid is used to transform an E.coli B strain of SE1 type [F-, CmR, ompT, Ion, hsdSB (rB-, mB-), gal, dcm, DE3 (lad, T7polymerase under the control of the PlacUV5 promoter). , ccdB +]. 2Apro of RVH-2 was produced by culturing the bacteria in a ZYP-5052 self-induction medium (Studier 2005) at 37 ° C for 5-6 hours with shaking, then at 20 ° C for 16-18 hours. . The cells are lysed with BugBuster (Novagen), centrifuged at 40000g for 15 min and the centrifugation supernatant obtained is subjected to a nickel resin affinity chromatography (HIS-Select ™ HF Nickel resin, Sigma). The 95% purified 2Apro is dialyzed against a buffer D (100 mM Tris-HCl, pH 7.5, 200 mM NaCl, 4 mM DTT) and concentrated on a Vivaspin (Sartorius Stedium Biotech) column. It is stored at -20 ° C. in buffer D containing 50% glycerol. The radioactive luciferases (13 μL) synthesized previously and containing the cleavage sites LVLQTM-GPSDM (SEQ ID No. 14) or PIITfAGPSDM (SEQ ID No. 16) are incubated for 45 min at 30 ° C. in the presence of 2 μg of RVH. Recombinant 2Apro (His) 6 and 13 μl of 2X reaction buffer (100mM HEPES / NaOH, pH 7.9, 200mM NaCl, 2mM EDTA, 10mM DU). Aliquots of the reaction mixture are taken 15 min, 30 min, 45 min and 1h after the start of the incubation. Proteins are separated by SDS-PAGE and analyzed by fluorography (Figure 3A).

Figure 3A shows that the luciferase containing the LVLQTMGPSDM site (SEQ ID NO: 14 - 61 kDa) is cleaved into two fragments of 25 kDa and 36 kDa in the first 15 minutes of the reaction with the same efficiency as observed for the site natural cleavage PIITTA-GPSDM (SEQ ID No. 16). Thus, these results demonstrate that the motif LVLQTM, SEQ ID No. 9 of RBM6 delta 6 behaves as an authentic half-site of the P6-P1 cleavage of the 2Apro of RVH-2, thereby presenting the expected characteristics for a inhibitor of 2Apro. 4) The peptide LVLQTM-GPSDM, SEQ ID No. 14 is cleaved by 2Apro ECHOvirus 6 According to the same strategy as that described above, the site cut LVLQTM-GPSDM (SEQ ID No. 14) by the 2Apro d ECHOvirus 6 (MGAFGQQSGAVYVGNYRVVNRHLATHTDWQNCVWEDYNRDLLVSTTTAHGCDTI ARCHCTSGVYFCASRNKHYPWFEGPGLVEVQESEYYPKRYQSHVLLAAGLSEPGDC GGILRCEHGVIGIVTMEGWGFADVRDLLWLEDDAMEQLEHHHHHH, SEQ ID NO: 17), another member of the enterovirus genus, has been studied. In these experiments, 2Apro of ECHO 6 is cloned into the vector pSCodon1.2 (Eurogentec) between the NdeI and XhoI restriction sites in fusion with a (His) tag on the C-terminal side. This plasmid is used to transform an E. coli strain B SE1 type and produce 2Apro ECHO 6 according to the culture and purification conditions described for the 2Apro RVH-2. The radioactive luciferases (13 μL) containing the LVLQTM-GPSDM (SEQ ID No. 14) or PIITTA-GPSDM (SEQ ID No. 16) cleavage sites are incubated for 45 min at 30 ° C. in the presence of 2 μg of 2Apro ( His) 6 purified ECHO 6 and 13 μl of 2X reaction buffer (100mM HEPES / NaOH, pH 7.9, 200mM NaCl, 2mM EDTA, 10mM DTT). Aliquots of the reaction mixture are taken 15 min, 30 min and 45 min after the start of incubation. Proteins are separated by SDS-PAGE and analyzed by fluorography (Figure 3B).

Figure 3B shows that ECHO 6A 2Apro has the same cleavage characteristics of the LVLQTM-GPSDM substrate (SEQ ID NO: 14) as the RVH-2 2Apro with complete digestion of the site in less than 15 min. This demonstrates that the LVLQTM motif (SEQ ID No. 9) is also recognized as an authentic half-site cleavage by 2Apro of ECHO 6. As the latter has a strong sequence homology with 2Apro other enteroviruses (poliovirus , Coxsackievirus), it is quite possible to apply these results to all 2Apro enteroviruses. 5) The VLQTM L peptide inhibits the cleavage activity of 2Apro of RVH-2 in vitro The hydrolysis of the colorimetric substrate TRPIITTA (SEQ ID No. 18) -pnitroanilide (SEQ ID No. 18-pNA) specific for 2Apro of RVH-2 was measured in vitro in the presence of the streptag-SUMO protein fused or not with the peptide LVLQTM, SEQ ID No. 9. This substrate contains the residues P6-SEQ ID No. 18-Pi of the VP1-2Apro cleavage site of the rhinovirus 2 polyprotein. The cleavage of this peptide between the alanine at the P1 position and the pNA releases the p-nitroaniline from yellow color whose absorbance is measured at 405 nm (Wang, Sommergruber et al., 1997). The activity test is carried out at 25 ° C in a final volume of 1 mL with a buffer containing 100 mM NaCl, 50 mM HEPES / NaOH pH8, 1 mM EDTA and 10 mM DU. Different concentrations of the StrepTag-SUMO-SEQ ID NO: 9 or StrepTag-SUMO recombinant proteins are pre-incubated with 0.2 μM of 2Apro of the RVH-2 for 5 min. Then 25 μM of substrate is added to initiate the reaction. Absorbance measurements are made continuously at 405 nm for 10 minutes to determine the initial rate of each reaction. The percent inhibition for a given StrepTag-SUMO-SEQ ID No. 9 peptide concentration was calculated by reporting the determined initial reaction rates in the presence and absence of peptide. The percentage inhibition values given in Figure 4 result from the average values obtained from 3 independent experiments. Standard deviations are also represented. In the presence of 25 μM substrate and 25 μM StrepTag-SUMO-SEQ ID No. 9 protein, the initial rate of the cleavage reaction is reduced by 80% relative to the control without peptide. The Streptag-SUMO protein has no effect on the reaction. Thus, these results thus indicate that the LVLQTM motif (SEQ ID No. 9) specifically and significantly inhibits the cleavage of SEQ ID No. 18-pNA by 2Apro of RVH-2 in vitro. In conclusion, the results presented previously show that: (i) the RBM6 delta 6 cellular protein has a C-terminal motif LVLQTM (SEQ ID NO: 9) necessary and sufficient to interact with 2Apro of RVH-2, (ii) This motif mimics the residue sequence at the P6-P1 positions of the 2Apro cleavage site of the RVH polyprotein and inhibits the cleavage activity of this enzyme, (iii) this motif is a pseudo-substrate of the RVH 2Apro. -2 but also of 2Apro of ECHO 6, the latter enzyme having strong homologies with 2Apro of other members of the same genus (Coxsackievirus, Poliovirus), (iv) IC50 of 10 pM measured for peptide LVLQTM (SEQ ID NO: 9) is much lower than that measured (600 μM) for peptides inhibiting poliovirus 2Apro (Ventoso, Barco et al., 1999) and substantially equal to that proposed for z.LSTT.fmk, a irreversible inhibitor of Coxsackievirus B3 2Apro (Badorff, Berkely et al., 2000). 6) The C-terminal fragment of RBM6 delta 6, RBM6 delta 6274 520, inhibits the cleavage activity of 2Apro of the RVH-2 in A549 cells In order to validate in cellulo the results obtained previously in vitro, the truncated form RBM6 delta 6274-520 identified in double hybrid was overexpressed in A549 cells (human lung epithelium) and the effects of this fragment on eIF-4G cleavage (eIF-4Gase activity) by 2Apro of RVH-2 were studied.

In this study, A549 cells are maintained in culture in DMEM medium (modified Dulbecco medium, BioWhittaker) supplemented with 10% (v / v) fetal calf serum, 2 mM L-glutamine, 100 U / mL penicillin and 100 μg / mL streptomycin, in an incubator at 37 ° C and in a saturated humidity atmosphere containing 5% CO 2. At confluence, the cells are detached from the support by treatment with trypsin-EDTA and reseeded in a culture flask containing 15 ml of fresh medium. On the day before transfection, A549 cells are seeded in 6 cm culture dishes (Corning) to reach 80% confluency at the time of transfection. The vectors pCiNeo [2xStreptag-RBM6 delta 6274-520] and / or p3xFlag [3xFlag-RVH-2 2Apro] are transfected using the NanoJuice reagents (Novagen) according to the manufacturer's recommendations. A control "transfection products alone" is performed. 1. medium with serum (complete medium) cells is replaced with serum-free medium (2 mM L-glutamine, 100 U / mL penicillin and 100 μg / mL streptomycin) prior to transfection 2. for each dish 6 cm, 300 μl of serum-free medium are mixed with 3 μl of "core reagent transfection" and 4.5 μl of "booster transfection" 3. incubation for 5 min at room temperature 4. addition of 5 μg of pCiNeo [2xStreptag-RBM6 delta 6274-524] and 3 μg of pCiNeo [2xStreptag] or 5 μg of p3xFlag [3xFlag-RVH-2 2Apro] and 3 μg of pCiNeo [2xStreptag] or 5 μg of p3xFlag [3xFlag-RVH-2 2Apro] and 3 μg pCiNeo [2xStreptag-RBM6 delta 6274-520] 5. incubation 15 min at room temperature 6. addition of the transfection mixture to the cells 7. incubation of the cells for 4 h at 37 ° C in a saturated humidity atmosphere containing 5% of CO2 8. the serum-free medium is replaced by complete medium 9. the cells are incubated at 37 ° C. in an atmosphere saturated with moisture The cellular proteins are extracted after 48 hours of transfection according to the following protocol: 1. the culture medium is sucked 2. the cells are rinsed twice with cold PBS 1X 3. the cells are incubated 15 min in ice with Mammalian Protein Extraction Reagent lysis solution (Roche) supplemented with a cocktail of protease inhibitors (Complete protease inhibitor Cocktail, Roche) (100 μL per box). 4. the whole is harvested in an eppendorf 1.5 mL 5. centrifugation 15 min 15000 xg at 4 ° C. 6. the supernatant is transferred to a new eppendorf The concentration of the proteins thus extracted is determined using the solution The Better Bradford Assay Kit (Pierce). 60 μg of protein from each sample are mixed with Laemmli 6X, heated for 5 min at 100 ° C. and then deposited for analysis on 6% acrylamide gel. After migration at 90 V for about 2 hours, the proteins are transferred to a nitrocellulose membrane for 1h30 to 400 mA. The membrane is then saturated with 5% milk (diluted in 1 × TBS) for 1 h at room temperature and then incubated overnight at 4 ° C. with an anti-eIF-4G antibody (rabbit polyclonal) diluted to 1000 ° in the solution. saturation. The membrane is incubated for 1 hour at room temperature with an anti-rabbit antibody coupled to peroxidase (GE Healthcare) diluted to 2000 in the saturation solution. The membrane is washed 4 times 20 min with TBS-tween (0.1%) and then subjected to analysis. Solutions 1 and 2 of the Pierce ECL Detection kit are used according to the instructions provided and the films (Amersham hyperfilm, GE Healthcare) are used to detect the signal. When RBM6 delta 6274-520 which contains the peptide LVLQTM, SEQ ID No. 9 and 2Apro of the RVH-2 are coexpressed in A549 cells, the eIF-4Gase activity of 2Apro is inhibited, as shown in FIG. 5, comparing the tracks RVH-2 2Apro and RVH-2 2Apro + RBM6 delta 6274.520. 7) The expression of the C-terminal fragment of RBM6 delta 6 (RBM6 delta 6274.520,) inhibits the infection of A549 cells by RVH-2 After having demonstrated that the truncated RBM6 delta form 6274-520 containing at its end C- terminating the sequence LVLQTM (SEQ ID NO: 9), inhibits the eIF-4Gase activity of 2Apro of RVH-2 in cellulo, the potential inhibitory effect of this fragment on the infectious cycle of RVH-2 in A549 cells. been studied. In this study, A549 cells are maintained in culture in DMEM medium (Dulbecco modified medium, BioWhittaker) supplemented with 10% (v / v) fetal calf serum, 2 mM L-glutamine, 100 U / mL penicillin and 100 μg / mL streptomycin. Incubation is performed at 37 ° C in a saturated humidity atmosphere containing 5% CO 2. At confluence, the cells are detached from the support by treatment with trypsin-EDTA and reseeded in a culture flask containing 15 ml of fresh medium. On the day before transfection, A549 cells are seeded in 6-well plates to reach 80% confluency at the time of transfection. Expression vectors pCiNeo [2xStreptag-RBM6 delta 6274-5201 or pCiNeo [2xStreptag-RBM6 delta 6274-514] or pCDNA3 [Streptag-SUMO] are transfected using Nano] uice reagents (Novagen) according to the manufacturer's recommendations. RBM6 delta 6274-514 corresponds to the truncated RBM6 delta form 6274.520 identified in double hybrid without the C-terminal sequence LVLQTM (SEQ ID No. 9). SUMO is a protein used here as a control. 1. medium with serum (complete medium) cells is replaced with serum-free medium (2 mM L-glutamine, 100 U / mL penicillin and 100 μg / mL streptomycin) prior to transfection 2. for each well. a 6-well plate, 200 μl of serum-free medium are mixed with 2 μl of "core reagent transfection" and 3 μl of "booster transfection" 3. incubation for 5 min at room temperature 4. addition of 4 μg of pCiNeo [ 2xStreptag-RBM6 delta 6274.520] or 4 μg of pCiNeo [Streptag-RBM6 delta 6274-514] or 4 μg of pCDNA3 [Streptag-SUMO] 5. incubation 15 min at room temperature 6. addition of the transfection mixture to the cells 7. incubation cells for 4h at 37 ° C in a saturated humidity atmosphere containing 5% CO 2 8. the serum-free medium is replaced by complete medium 9. the cells are incubated at 37 ° C in a saturated humidity atmosphere containing 5% The cells are infected with RVH-2 at a MOI 1, that is, in theory, each cell is infected with a viral particle after 24 hours of transfection according to the following protocol: 1. the culture medium is aspirated 2. the cells are rinsed once with DMEM medium 2% serum, 2 mM of L-glutamine, 100 U / mL of penicillin and 100 μg / mL of streptomycin 3. the cells are inoculated with the virus and 1.5 mL of DMEM medium 2% serum are added 4. the cells thus inoculated are incubated at 34 The supernatant of the infected cells is removed 20 hours after infection with RVH-2. Supernatants are titrated on MRC5 cells and TCID50 is determined by the method of Reed and Muench (Reed LJ 1938) according to the following protocol: 1. MRC5 cells are maintained in culture in EMEM medium 2% serum, 2 mM of L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin and 2% 1M hepes and seeded in 24-well plates 5 days before inoculation with RVH-2. They are confluent on the day of infection. 2. On the day of infection, eight 10-fold dilutions of 10-1 to 10-8 are made for each sample so as to inoculate 100 μL of each 6-well dilution per 24-well plate. 3. The cell survival medium (1.4 mL) is changed before distribution of plaque viral suspensions. 4. 100 μl undiluted viral suspension is distributed on 6 wells of 24-well plates. 5. In the same way, 100 μl of each virus dilution is distributed over 6 wells of 24-well plates. Several virus-free cookies are also made. 6.The plates are incubated at 34 ° C for 5 days. 7. On day 5, TCID50 is calculated using the method of Reed and Muench.

This experiment was repeated 3 times.

In cells overexpressing RBM6 delta 6274-520 (fragment which contains at its C-terminus the peptide LVLQTM, SEQ ID No. 9), the viral titer reaches 105'5 TCID 50 / mL whereas for cells overexpressing the control protein (SUMO), the resulting titre of 10''5 TCID50 / mL, is approximately 100-fold higher. RBM6 delta 6274-520 reduces the production of RVH-2 viruses in A549 cells. In cells overexpressing RBM6 delta 6274-514 (fragment which does not contain at its C-terminus the peptide LVLQTM, SEQ ID No. 9), the viral titer obtained from 1073 TCID50 / mL demonstrates that the sequence LVLQTM SEQ ID N ° 9 located at the C-terminus of RBM6 delta 6274-520 is responsible for the decrease in viral production in A549 cells. Badorff, C., N. Berkely, et al. (2000). "Enteroviral protease 2A directly cleaves dystrophin and is inhibited by a similar dystrophin-based substrate." Biol Chem 275 (15): 11191-11197. Deszcz, L., R. Cencic, et al. (2006). "An antiviral peptide inhibitor that is active against picornavirus 2A proteinases but not cellular caspases." Virol 80 (19): 9619-9627.

Deszcz, L., J. Seipelt, et al. (2004). "Antiviral activity of caspase inhibitors: effect on picornaviral 2A proteinase." FEBS Lett 560 (1-3): 51-55. Formstecher, E., S. Aresta, et al. (2005). "Protein interaction mapping: a Drosophila case study." Genome Res 15 (3): 376-384.

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Claims (17)

CLAIMS1 - Peptides whose C-terminal part corresponds to the sequence: X1-X2-X3-X4-X5-X6 in which XI and X3, identical or different, are Leu, Ile or Phe, or an unconventional amino acid derived from Leu, Ile or Phe, X2 and X4, identical or different, are any amino acid, - X5 is Asn, Thr or Gln, or an unconventional amino acid derived from Asn, Thr or Gln, and - X6 is an amino acid hydrophobic, as well as the chemical derivatives of such peptides, with the exception of the protein RBM6 delta 6. 2 - Peptides according to claim 1 characterized in that their C-terminal portion corresponds to the sequence: X1-X2-X3-X4-X5-X6 wherein: X1 and X3, identical or different, are Leu, Ile or Phe, preferably Leu or Ile, - X2 and X4, identical or different, are any amino acid, - X5 is Asn, Thr or Gln, preferably Asn or Thr, and - X6 is a hydrophobic amino acid, as well as chemical derivatives such peptides, with the exception of RBM6 delta 6 protein. 3 - Peptides according to claim 1 or 2 characterized in that they correspond to the X1-X2-X3-X4-X5-X6 sequence as defined in claim 1 or 2, as well as the chemical derivatives of such peptides. 4 - Peptides according to one of the preceding claims characterized in that X6 is an amino acid selected from Leu, Cys, Val, Met, Pro, Tyr, Ile, Ala, Phe, Trp and unconventional amino acids derived from Leu, Cys, Val, Met, Pro, Tyr, Island, Ala, Phe, and Trp. 5 - Peptides according to one of the preceding claims characterized in that X6 is an amino acid selected from Leu, Cys, Val, Met, and preferably is Met. 6 - Peptides according to one of the preceding claims characterized in that X2 and X4 identical or different, are an amino acid selected from Val, Irregularity Notification Response 26 August 2 9992 32 Lys, Sern Thr, Arg, Cys, His, Phe, Tyr and Gin and the unconventional amino acids derived from these. 7 - Peptides according to one of the preceding claims, characterized in that their C-terminal part corresponds to a hexapeptide chosen from: IKLVTL (SEQ ID NO: 2), LSLVTL (SEQ ID NO: 3), FRLTTL (SEQ ID No. 4), LCLHTC (SEQ ID NO: 5), LFLYTC (SEQ ID NO: 6), LYIYTV (SEQ ID NO: 7), LKLQTV (SEQ ID NO: 8), LVLQTM (SEQ ID NO: 9), LRLKNL (SEQ ID NO: 10), LRLRNL (SEQ ID NO: 11), and preferably hexapeptide LVLQTM (SEQ ID NO: 9). 10 8 - Peptides according to one of the preceding claims characterized in that they are chosen from: IKLVTL (SEQ ID No. 2), LSLVTL (SEQ ID No. 3), FRLTTL (SEQ ID No. 4), LCLHTC (SEQ ID NO: 5), LFLYTC (SEQ ID NO: 6), LYIYTV (SEQ ID NO: 7), LKLQTV (SEQ ID NO: 8), LVLQTM (SEQ ID NO: 9), LRLKNL (SEQ ID NO: 10) , LRLRNL (SEQ ID NO: 11) and preferably is the hexapeptide LVLQTM (SEQ ID NO: 9), as well as their chemical derivatives. 9 - A peptide selected from the peptides according to one of the preceding claims and the isolated RBM6 delta 6 protein, for use in the therapeutic or prophylactic treatment of enterovirus. Peptide according to one of claims 1 to 9 as a medicament for the therapeutic or prophylactic treatment of a condition selected from colds, otitis, sinusitis, acute flaccid paralysis, aseptic meningitis, hand-foot-and-mouth syndrome. , respiratory diseases, acute or chronic heart diseases, diarrhea, pancreatitis, ocular lesions and encephalitis. 25 11 - Peptide according to one of claims 1 to 9 for its use in the therapeutic or prophylactic treatment of a rhinovirus, and in particular a human rhinovirus such as RVH-2, or an ECHOvirus. 12 - Peptide according to one of claims 1 to 11 characterized in that it has an inhibitory activity of a cellular or viral protease. 13 - Peptide according to claim 12 characterized in that it has an inhibitory activity of the protease 2A Human Rhinovirus, and in particular the RVH-2. Answer to irregularities notification of 26th August 2 (0 "5 9992 33 14 - Peptide according to claim 12 characterized in that it has an inhibitory activity of a cellular or viral protease, other than protease 2A rhinovirus. A pharmaceutical composition comprising a peptide selected from the peptides according to one of claims 1 to 14, in association with a pharmaceutically acceptable excipient. 16 - Composition according to claim 15 for the treatment of an Enterovirus, and in particular Rhinovirus or ECHOvirus. 17 - Use of a peptide according to one of claims 1 to 8 and 12 to 14, for the preparation of cell lysates.